Biophysical, sequence and structural analysis of type 3 secretion system pilus protein HrpA of : insights into HrpA pili stability and assembly.

Type 3 secretion system (T3SS) is an essential virulence system utilized by many gram-negative bacteria including to deliver effector proteins into host cells. The extracellular, long, needle-like proteinaceous complex (pilus) of T3SS transports effectors. In , HrpA, an 11 kDa protein assembles to form the pilus structure, whose structure and stability remain poorly understood. To address this, recombinant HrpA protein was prepared and carried out the biophysical characterization. The native PAGE and dynamic light scattering analysis showed higher-order oligomerization of rHrpA with hydrodynamic radii in the range of 1-1000 nm. Transmission Electron Microscopy revealed that rHrpA spontaneously forms needle-like filaments (∼8 nm in width and ∼129-300 nm in length) and also the aggregation of the filaments. CD spectroscopic analysis showed the predominantly helical nature of rHrpA. We examined the effects of detergents, denaturants, pH and temperature on rHrpA assemblies. Detergents such as SDS and sarkosyl effectively disrupted the oligomers, whereas urea and guanidine hydrochloride had no effect. The sequence analysis of HrpA from different pathovars of revealed that, for the first time, two groups of HrpA proteins (108 aa and 113 aa) that are sequence-wise unrelated. Structural comparison of HrpA with animal filament proteins such as MxiH, PrgI and BsaL revealed that plant pilus structures could be highly flexible compared to animal filaments. This study contributes to our understanding of the T3SS pilus structure and its stability. The results of this study could lead to new approaches for T3SS pilus protein structure-function investigation.